PDBsum entry 2ok4

Oxidoreductase

PDB id: 2ok4

Contents

Protein chains

110 a.a. *

122 a.a. *

360 a.a. *

Ligands

HY1 ×2

Waters ×1051

* Residue conservation analysis

PDB id:

2ok4

Name:

Oxidoreductase

Title:

Crystal structure of aromatic amine dehydrogenase ttq- phenylacetaldehyde adduct oxidized with ferricyanide

Structure:

Aromatic amine dehydrogenase, small subunit. Chain: d, h. Fragment: (residues: 48-182). Aromatic amine dehydrogenase, large subunit. Chain: a, b. Fragment: (residues: 73-433). Ec: 1.4.99.4

Source:

Alcaligenes faecalis. Organism_taxid: 511. Organism_taxid: 511

Resolution:

1.45Å R-factor: 0.156 R-free: 0.188

Authors:

A.Roujeinikova,D.Leys

Key ref:

A.Roujeinikova et al. (2007). New insights into the reductive half-reaction mechanism of aromatic amine dehydrogenase revealed by reaction with carbinolamine substrates. J Biol Chem, 282, 23766-23777. PubMed id: 17475620 DOI: 10.1074/jbc.M700677200

Date:

16-Jan-07 Release date: 01-May-07

Protein chain

P84887  (AAUA_ALCFA) -  Aralkylamine dehydrogenase light chain from Alcaligenes faecalis

Seq: 182 a.a.

Struc: 110 a.a.*

Protein chain

P84887  (AAUA_ALCFA) -  Aralkylamine dehydrogenase light chain from Alcaligenes faecalis

Seq: 182 a.a.

Struc: 122 a.a.*

Protein chains

P84888  (AAUB_ALCFA) -  Aralkylamine dehydrogenase heavy chain from Alcaligenes faecalis

Seq: 390 a.a.

Struc: 360 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chains D, H, A, B: E.C.1.4.9.2 - aralkylamine dehydrogenase (azurin).
• Reaction: an aralkylamine + 2 oxidized [azurin] + H2O = an aromatic aldehyde + 2 reduced [azurin] + NH4⁺ + 2 H⁺

aralkylamine + 2 × oxidized [azurin] + H2O = aromatic aldehyde (Bound ligand (Het Group name = HY1) matches with 88.89% similarity) + 2 × reduced [azurin] + NH4(+) + 2 × H(+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1074/jbc.M700677200

J Biol Chem 282:23766-23777 (2007)

PubMed id: 17475620

New insights into the reductive half-reaction mechanism of aromatic amine dehydrogenase revealed by reaction with carbinolamine substrates.

A.Roujeinikova, P.Hothi, L.Masgrau, M.J.Sutcliffe, N.S.Scrutton, D.Leys.

ABSTRACT

Aromatic amine dehydrogenase uses a tryptophan tryptophylquinone (TTQ) cofactor to oxidatively deaminate primary aromatic amines. In the reductive half-reaction, a proton is transferred from the substrate C1 to betaAsp-128 O-2, in a reaction that proceeds by H-tunneling. Using solution studies, kinetic crystallography, and computational simulation we show that the mechanism of oxidation of aromatic carbinolamines is similar to amine oxidation, but that carbinolamine oxidation occurs at a substantially reduced rate. This has enabled us to determine for the first time the structure of the intermediate prior to the H-transfer/reduction step. The proton-betaAsp-128 O-2 distance is approximately 3.7A, in contrast to the distance of approximately 2.7A predicted for the intermediate formed with the corresponding primary amine substrate. This difference of approximately 1.0 A is due to an unexpected conformation of the substrate moiety, which is supported by molecular dynamic simulations and reflected in the approximately 10(7)-fold slower TTQ reduction rate with phenylaminoethanol compared with that with primary amines. A water molecule is observed near TTQ C-6 and is likely derived from the collapse of the preceding carbinolamine TTQ-adduct. We suggest this water molecule is involved in consecutive proton transfers following TTQ reduction, and is ultimately repositioned near the TTQ O-7 concomitant with protein rearrangement. For all carbinolamines tested, highly stable amide-TTQ adducts are formed following proton abstraction and TTQ reduction. Slow hydrolysis of the amide occurs after, rather than prior to, TTQ oxidation and leads ultimately to a carboxylic acid product.

Selected figure(s)

Figure 3.
FIGURE 3. Schematic overview of the proposed mechanism for AADH R-carbinolamine oxidation. For ease of comparison with the previously proposed amine oxidation mechanism, a similar notation is used according to Fig. 1. For clarity, only part of the TTQ cofactor is represented, whereas the side chain of the different R-carbinolamines is indicated by an R. Refer to supplementary Scheme 1 for a full description of the substrate-TTQ-enzyme adduct. The active site water molecule (or ammonia in case of a steady state mechanism, see Ref. 24) is denoted W1. Whether a conformational equilibrium between IIIb-A and IIIb-B occurs depends on the nature of the R side chain.

Figure 5.
FIGURE 5. Top, comparison of IIIb-A conformation as observed in the crystals (phenylacetaldehyde derived carbon atoms in cyan) with a more optimal configuration (IIIb-B) based on the modeled intermediate IIIa (Fig. 1) during tryptamine reduction (Ref. 8; phenylacetaldehyde-derived carbon atoms in yellow). Putative hydrogen bonding interactions made between IIIb-B conformation and active site residues are shown by dotted lines. Key active site residues and TTQ cofactor are displayed with green carbons. B, overlay of crystal structures of IIIb-A (with green carbons) and Vc (with cyan carbons) for phenylacetaldehyde and ammonia as substrates. The active site water molecule situated close to C-6 and O-7 is shown as a red sphere (labeled W-1[ox]and W-1[red], respectively).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 282, 23766-23777) copyright 2007.

Figures were selected by an automated process.